Heavy metal ion induction of adhesion molecules and cytokines in human endothelial cells: the role of NF-kappaB, I kappaB-alpha and AP-1.
We analyzed the influence of heavy-metal ions on human umbilical vein endothelial cells (HUVEC) in comparison to proinflammatory cytokines (TNF-alpha, IL-1beta) and lipopolysaccharide (LPS). Adhesion molecule and cytokine expressions are upregulated by heavy-metal exposure. Expression of E-selectin on the cell surface was strongly induced by 1-mM concentrations of NiCl2 and CoCl2, whereas ZnCl2 and CrCl3 had no influence. Furthermore, it is shown that NiCl2 induces mRNA expression of E-selectin, intercellular adhesion molecule-1, IL-6 and IL-8 in a 1-mM concentration. The transcription factor NF-kappaB is known to be involved in the regulation of adhesion molecule expression in endothelial cells after activation by proinflammatory cytokines. We demonstrated that treatment of HUVEC with Ni2+ and Co2+ ions induces the translocation of NF-kappaB p65 and also p50 into the nucleus. NF-kappaB binding activity is enhanced under the influence of heavy metals as determined by mobility shift analysis. P65 and p50 are components of the NF-kappaB complexes as confirmed by supershift analysis. We could show that activation at the protein level is accompanied by induction of NF-kappaB p65 mRNA expression. HUVEC also express the NF-kappaB inhibitor I kappaB-alpha (MAD-3). In the early phase of activation by Ni2+ and Co2+ ions, disappearance of I kappaB-alpha in the cytoplasm accompanied p65 translocation, followed by its gradual reappearence. Because I kappaB mRNA could be upregulated by NiCl2 as well as by a mixture of cytokines, we suggest that the replenishment of the inhibitor in the cytoplasm is caused by de novo I kappaB gene expression. In addition to the enhanced DNA-binding activity of NF-kappaB, another transcription factor, AP-1, was also augmented in HUVEC stimulated by NiCl2, CoCl2 or by proinflammatory mediators and the phorbol ester PMA. Fos protein is shown to be a component of the activated AP-1 complex, as determined by supershift analysis, suggesting that it consists of Jun/Fos heterodimers.
Wagner
M
M
Department of Pathology, Johannes Gutenberg University, Mainz, Germany.
Klein
C L
CL
Kleinert
H
H
Euchenhofer
C
C
Förstermann
U
U
Kirkpatrick
C J
CJ
D015152:Blotting, Northern; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D015342:DNA Probes / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins; D019216:Metals, Heavy / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins; D015854:Up-Regulation
D015815:Cell Adhesion Molecules; D016207:Cytokines; D015342:DNA Probes; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D019216:Metals, Heavy; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D000072000:NF-KappaB Inhibitor alpha; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013755:Tetradecanoylphorbol Acetate